Five spin-labeled 9-aminoacridines, each bearing either a 4-(2,2,6,6-tetramethyl-1-piperidinyloxy) or a 3-(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety in the 9-position, have been synthesized and assayed for biological activity in three different test systems. Sedimentation velocity measurements indicated that the labels caused unwinding of calf thymus DNA. Those acridines which contained both 6-chloro and 2-methoxy substituents were less toxic to leukemia L1210 cells in static culture than the corresponding unsubstituted analogs. While the unsubstituted aminoacridines were quite good inhibitors of E. coli DNA-primed RNA polymerase, the 6-chloro-2-methoxy substituted compounds stimulated this enzyme system. In the presence of E. coli DNA, the electron spin resonance spectrum of 4-(6-chloro-2-methoxy-9-acidinyl) amino)-2,2,6,6-tetramethyl-1-piperidinyloxy became broad and asymmetric with a maximal hyperfine splitting (2T") of 57.5 gauss. This finding indicates that when the acridine spin label intercalates into DNA the piperidinyl moiety that bears the nitroxide group becomes highly immobilized. These results suggest that the spin-labeled 9-aminoacridines will be useful probes for nucleic acids.